Molecular immobilization is one of the major technologies for life science research. Accordingly, both traditional technologies, such as hybridization, and recently developed technologies, such as biological chips, microarrays and microfluidic systems, play a key role in the bioanalysis. Generally speaking, there are five types of molecular immobilization classified for life science research: physical adsorption, ionic binding, covalent binding, cross-linking binding, and entrapping.
In physical adsorption technologies, the biospecimen is applied on a carrier by using hydrophobic interactions, hydrophilic interactions, hydrogen binding, and affinity. The advantage of physical adsorption technologies is their ease of operation, low operation cost, and relatively small impact on the integrity of the biospecimen. However, the disadvantage of physical adsorption technologies is that the immobilized biospecimen can be desorbed due to the environmental conditions such as temperature or pH.
Ionic binding technologies make use of opposite electric charges between the carrier and the biospecimen. These opposite electric charges result in electrostatic interactions between the biospecimen and the carrier that cause the biospecimen to become immobilized on the carrier. The advantages of ionic binding technologies are their ease of operation and the ability to stably fix the biospecimens on the carrier. However, the disadvantage of the ionic bonding technologies is that the molecular immobilization can become desorbed due to environmental conditions such as temperature and pH.
Covalent binding technologies make use of covalent binding between the carrier and the biospecimen to fix the biospecimen onto the carrier. Many biospecimens carry a reactive functional group. Representative reactive functional groups include, but are not limited to carboxyls, hydroxyls, aminos, and/or thiols as well as photosensitive groups (e.g. aryl azides, halogenated aryl azides, benzophenones, diazos, and diazierine derivatives). See, for example, Hermanson, Bioconjugate Techniques, Academic Press; 1 st edition (Jan. 15, 1996). Such functional groups can be used to form the covalent binding with the carrier thereby causing the biospecimen to become substantially immobilized on the carrier. Unlike the physical adsorption technology and the ionic binding technology, the covalent binding between the carrier and the bio-specimen is not influenced by environmental conditions such as temperature or pH. In addition, depending on the nature of the biospecimen, the carrier can be made of a wide variety of different materials, such as glass, metal, nature polymer, or artificial polymer. However, an intermediate layer, such as a coupling agent or linker, is often utilized to immobilize the biospecimen on the carrier when the bio-specimen does not carry the functional group. Examples of covalent binding technologies are found, for example, in U.S. Patent Publication Nos. 20020049152 and 20040058390 to Nock et al.
Cross-linking binding technologies use multi-functional groups as cross-linkers. Such compounds have a plurality of amino, thiol, carbonyl, and/or carboxyl reactive groups that form a network of inter-molecular interactions between the carrier and the biospecimen thereby causing the biospecimen to become attached to the carrier. Representative cross-linkers include zero-length cross-linkers (e.g., the carbodiimides EC, EDC plus sulfo-NHS, CMC, DCC, and DIC; Woodwards Reagent K; N,N′-carbonyldiimidazole, and the use of Schiff base formation coupled with reductive amination), homobifunctional cross-linkers (e.g., the homobifunctional NHS esters DSP, DTSSP, DSS, BS, DST, sulfo-DST, BSOCOES and sulfo-BSOCOES, EGS and sulfo-EGS, DSG, and DSC; the homobifunctional imidoesters DMA, DMP, DMS and DTBP; the homobifunctional sulfhydryl-reactive crosslinkers DPDPB and BMH; the difluorobenzene derivatives DFDNB and DFDNPS; the homobifunctional photoreactive crosslinker BASED; the homobifunctional aldehydes formaldehyde and glutaraldehyde; the bis-epoxides 1,4-butanediol diglycidyl ether; the homobifunctional hydrazines adipic acid dihydrazide and carbohydrazide; bis-diazoniaum derivatives and bis-alkylhalides) and trifunctional crosslinkers (e.g., 4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester and sulfo-SBED). See, for example, Hermanson, 1996, Bioconjugate Techniques, Academic Press. Accordingly, a cross-linker is adapted to enhance the strength of the molecular immobilization on the carrier. However, the cross-linking binding technology may cause the denaturalization of the biospecimen.
Entrapment-type molecular immobilization technologies dispose the biomaterial on a natural or artificial polymer, such as agarose or polyacrylamide. Such polymers contain a plurality of pores each having a predetermined size for diffusion. Thus, the biospecimen becomes entrapped in these pores.
The above-described protein immobilization technologies are used for bioassays. A popular bioassay immobilization technology for use in the study of bioreactions is nitrocellulose paper. Nitrocellulose paper, which is made of a nitrocellulose polymer, has a thickness of approximately 150 micrometers and has a pore size that allows for control over the bioreaction. Nitrocellulose has a relatively strong affinity to DNA, RNA nd protein. Thus, nitrocellulose is considered to be a suitable material for the analysis of DNA, RNA, and protein because of the low cost and purity of the material. In addition, nitrocellulose prevents the denaturization of the biospecimen. Moreover, the use of nitrocellulose is relatively inexpensive because the steps required to react the biospecimen with the nitrocellulose are simple. Nitrocellulose having pore sizes anywhere in a broad range of pore sizes can be made. The diversity in pore sizes that can be used provides considerable control over the bioreaction that are run on nitrocellulose. Furthermore, nitrocellulose minimizes the environmental pollution because it is not a poisonous chemical material like those that are often used in entrapment-type molecular immobilization technologies.
In some conventional nitrocellulose molecular immobilization protocols, the nitrocellulose is applied on a glass slide by cutting the nitrocellulose paper into a desired size corresponding to the glass slide. The nitrocellulose paper can be purchased, for example, from Schleicher and Schuell (Protran Nitrocellulose Transfer Membranes, USA) or Sigma-Aldrich, USA (catalog numbers N7892, N8017, N8142, N8267, and N8392). The nitrocellulose paper is then affixed to the glass slide. Alternatively, a nitrocellulose coating is formed either by submerging the glass slide into the nitrocellulose solution or by applying the nitrocellulose solution on the glass slide by dropwise addition.
European Patent Application EP-0366241 to Brigati (“Brigati”) discloses one method of forming a nitrocellulose media suitable for binding reactions. In Brigati, slides were rinsed with distilled water and then dipped in a two percent solution of APTS (Cat. No. A3648 from Sigma Chemical) in distilled water for two minutes. The slides were then rinsed by dipping in five successive one liter vessels of distilled water and dried in an 80° C. convection oven and then at room temperature in air. The slides with positively charged surfaces made by such a treatment were then dipped into conical 50 mL centrifuge tubes containing a solution of nitrocellulose in methanol. That solution had been made by cutting up two nitrocellulose membranes (88 mm by 88 mm Schleicher and Schuell BA-85) of a total weight of about 1.1 g and dissolving the pieces in a total of 30 ml of absolute methanol by rotary stirring. The slides removed from the centrifuge tubes were then dried in a horizontal orientation in a microwave oven for thirty seconds. This resulted in a clear layer of nitrocellulose having a thickness of approximately 5-10 μm thickness coating the slides glass and epoxy surfaces that had been immersed in the methanol solution. Thickness of the coating was adjusted by applying additional layers of nitrocellulose/methanol solution to the first application. The drawback of the Brigati method is that it requires multiple steps, particularly when a nitrocellulose layer having a thickness greater than 10 μm is desired.
The microassay is an important tool in many existing technologies. However, the use of nitrocellulose coating for microassay analysis, such as found in microfluidic systems, is limited by the physical properties of nitrocellulose. Such limiting properties include the difficulty in controlling nitrocellulose thickness and the complicated manufacturing process used to make nitrocellulose-based assays. Such drawbacks limit the utility of conventional nitrocellulose coatings in existing bioassays. Thus, given the above background, what are needed in the art are improved systems and methods for manufacturing nitrocellulose-based assays.